Therapeutic composition including phenolic compounds derived from Opuntia littoralis

ABSTRACT

A therapeutic composition including phenolic compounds derived from Opuntia littoralis (OL) includes an extract of Opuntia littoralis and lutein. The Opuntia littoralis extract can be formed by extracting an Opuntia littoralis plant powder with an alcohol to provide an Opuntia littoralis alcohol extract (OLAE) including the phenolic compounds. In an embodiment, the therapeutic composition can be effectively used as an analgesic for the treatment of pain, as an anti-inflammatory, as a treatment for immunological disorders, and/or as a treatment for oxidant conditions.

BACKGROUND 1. Field

The disclosure of the present patent application relates to antioxidantand anti-inflammatory compositions and, more particularly, tocompositions including phenolic compounds derived from Opuntialittoralis.

2. Description of the Related Art

Therapeutic preparations which are based on natural ingredients canfrequently provide relief for several ailments at once. Differentspecies of plants, for example, contain nutrients and compounds that areuseful for various human health issues. For this reason, many plants aretypically used for treating cosmetic or medical conditions. Cactiplants, in particular, are considered a rich source of a variety ofbeneficial nutrients and compounds.

Opuntia littoralis is a species of prickly pear cactus known by thecommon name coastal prickly pear. Prickly pear is high in fiber,antioxidants, and carotenoids. The edible parts of prickly pear includethe fruit, stems, leaves, and flowers.

Thus, a therapeutic composition including phenolic compounds derivedfrom Opuntia littoralis solving the aforementioned problems is desired.

SUMMARY

A therapeutic composition including phenolic compounds derived fromOpuntia littoralis (OL) includes an alcoholic extract of Opuntialittoralis and lutein. The Opuntia littoralis extract can be formed byextracting an Opuntia littoralis plant powder with an alcohol to providean Opuntia littoralis alcohol extract (OLAE) including the phenoliccompounds. In an embodiment, the therapeutic composition can beeffectively used as an analgesic for the treatment of pain and asanti-inflammatory. In an embodiment, the therapeutic composition can beuseful for treating immunological disorders. In an embodiment, thetherapeutic composition can provide therapeutic use in the treatment ofoxidant conditions.

A pharmaceutical composition can include a therapeutically effectiveamount of the therapeutic composition and a pharmaceutically acceptablecarrier or excipient. In some embodiments, the present compositions canbe used for combination therapy, where other therapeutic and/orprophylactic ingredients can be included therein.

A process for the preparation of the therapeutic composition can includeproviding a plant part of OL, reducing the plant part to a powder, andextracting the OL with water and alcohol to obtain an OL alcohol extract(OLAE), which comprises the phenolic acids and flavonoids of OLmolecules. The plant powder can be formed by drying the Opuntialittoralis plant or plant part, e.g., cladode and fruits, and reducingthe dried plant or plant part to a powder. The plant powder can beextracted from the part of the Opuntia littoralis plant with 70% aqueousethanol over multiple days, for example, 8 days. The OLAE can be mixedwith an emulsified lutein homogenate to provide an emulsion.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are graphs showing oxidative stress in LPS induced RAW 264.7cell lines estimated based on the activity of MPO in μM/mg of (FIG. 1A)OLAE-treated cells; (FIG. 1B) lutein-treated cells; and (FIG. 1C) OLAEand lutein-treated cells.

FIG. 2 is a graph showing cytotoxicity measured by SRB assay of O.littoralis alcohol extract OLAE (25 μg/mL), Lut (2.5 μM) treatment, andthe therapeutic composition according to the present teachings.

FIG. 3 is a graph shows Nitric Oxide (NO) in OLAE-treated,lutein-treated, and OLAE and lutein-treated limb tissue.

FIG. 4 is a graph showing cell invasion assessed using transwell cellculture Boyden chambers seeded with OLAE (25 μg/mL), lutein (2.5 μM),OLAE plus lutein, and 100 μL of MDA cells (5×10⁴).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions are provided for the purpose of understandingthe present subject matter and for construing the appended patentclaims.

It should be understood that the drawings described above or below arefor illustration purposes only. The drawings are not necessarily toscale, with emphasis generally being placed upon illustrating theprinciples of the present teachings. The drawings are not intended tolimit the scope of the present teachings in any way.

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

“Subject” as used herein refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, and petcompanion animals such as household pets and other domesticated animalssuch as, but not limited to, cattle, sheep, ferrets, swine, horses,poultry, rabbits, goats, dogs, cats and the like.

“Patient” as used herein refers to a subject in need of treatment of acondition, disorder, or disease, such as an inflammatory condition or animmunological disorder.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise. As used herein, the term “about” refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

The present disclosure is related to a therapeutic composition includingphenolic compounds derived from Opuntia littoralis (OL). In anembodiment, the therapeutic composition includes an alcoholic extract ofOpuntia littoralis and lutein. In an embodiment, the therapeuticcomposition can include from about 0.1 gram to about 1 gram of Opuntialittoralis and about 1 mM to about 10 mM lutein. The therapeuticcomposition can be effectively used to treat at least one conditionselected from pain, inflammation, an immunological disorder, and anoxidant condition.

In an embodiment, the therapeutic composition can include an Opuntialittoralis alcohol extract (OLAE). The OLAE can be prepared by preparinga plant powder from a part of the Opuntia littoralis plant. In anembodiment, the part of the Opuntia littoralis plant or plant partincludes at least one of a fruit and a cladode of the Opuntia littoralisplant. The plant part can be dried and reduced to a plant powder. Theplant powder can be extracted with an alcohol such as ethanol. In anembodiment, the plant powder can be combined with 70% aqueous ethanolover multiple days to obtain the OLAE. In certain embodiments, the plantpowder can be combined with the aqueous ethanol once, twice, threetimes, four times, five times, or more for a period of two, three, four,five, six, seven, eight, nine, ten, or more days to provide a residue.In an embodiment, the plant powder can be combined with the aqueousethanol four times for a period of about eight days to provide aresidue. Any inorganic salts and non-phenolic compounds can be removedfrom the residue to provide the OLAE.

According to an embodiment, a method of preparing the therapeuticcomposition can include combining the extract of Opuntia littoralis withan emulsified lutein homogenate. In an embodiment, the extract ofOpuntia littoralis is OLAE. In an embodiment, the emulsified luteinhomogenate can be prepared by mixing a lutein powder with stearic acid.

A process for the preparation of OL phenolic compounds can includeproviding a plant part of OL, reducing the plant part to a powder, andextracting the OL with water and alcohol to obtain an OL alcohol extract(OLAE). The OLAE can be mixed with an emulsified lutein homogenate toprovide an emulsion including the phenolic acids and flavonoids of OL.In an embodiment, the present subject matter relates to a therapeuticcomposition produced according to the present synthesis methods.

The therapeutic composition can have more than one therapeutic use. Inan embodiment, the therapeutic composition can be effectively used as ananalgesic for the treatment of pain. In an embodiment, the therapeuticcomposition can be effectively used as an anti-inflammatory. In anembodiment, the therapeutic composition can be useful for treatingimmunological disorders. In an embodiment, the therapeutic compositionprovides antioxidant activity as well as therapeutic use in thetreatment and management of pain and inflammatory conditions.

In some embodiments, the present compositions can be used forcombination therapy, where other therapeutic and/or prophylacticingredients can be co-administered therewith.

A pharmaceutical composition can include the therapeutic composition anda pharmaceutically acceptable carrier or excipient. Non-limitingexamples of suitable excipients, carriers, or vehicles useful hereininclude liquids such as water, saline, glycerol, polyethyleneglycol,hyaluronic acid, ethanol, and the like. Suitable excipients fornonliquid formulations are also known to those of skill in the art. Inan embodiment, a therapeutically effective amount of the therapeuticcomposition can include from about 0.01 mg to 500 mg per unit dose. Forexample, a therapeutically effective amount of the therapeuticcomposition can include from about 1 mg to about 100 mg per unit dose.

The present compounds are typically administered at a therapeutically orpharmaceutically effective dosage, e.g., a dosage sufficient to providetreatment for an immunological disease or an inflammatory disorder.Administration of the compounds or pharmaceutical compositions thereofcan be by any method that delivers the compounds systemically and/orlocally. These methods include oral routes, parenteral routes,intraduodenal routes, and the like.

While human dosage levels have yet to be optimized for the presentcompounds, generally, the pharmaceutical composition can include about0.01 mg to 500 mg of the therapeutic composition per unit dose, forexample about 1 mg to about 100 mg per unit dose. The precise effectiveamount will vary from subject to subject and will depend upon thespecies, age, the subject's size and health, the nature and extent ofthe condition being treated, recommendations of the treating physician,and the therapeutics or combination of therapeutics selected foradministration. The subject may be administered as many doses as isrequired to reduce and/or alleviate the signs, symptoms, or causes ofthe disease or disorder in question, or bring about any other desiredalteration of a biological system.

In employing the present composition for treatment of pain, immunedisorders, oxidant conditions, and/or inflammatory conditions, anypharmaceutically acceptable mode of administration can be used withother pharmaceutically acceptable excipients, including solid,semi-solid, liquid or aerosol dosage forms, such as, for example,tablets, capsules, powders, liquids, suspensions, suppositories,aerosols or the like. The present compounds can also be administered inimmediate, sustained or controlled release dosage forms, including depotinjections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.

The present compounds may also be administered as compositions preparedas a “dietary supplement” or a product that is intended to supplementthe human diet and may be provided in the form of a pill, capsule,tablet, or like formulation. By way of non-limiting example, a dietarysupplement may include one or more of the following dietary ingredients:vitamins, minerals, herbs, botanicals, amino acids, and dietarysubstances intended to supplement the diet by increasing total dietaryintake, or a concentrate, metabolite, constituent, extract, orcombinations of these ingredients, not intended as a conventional foodor as the sole item of a meal or diet. Dietary supplements may also beincorporated into foodstuffs, such as functional foods designed topromote control of glucose levels. A “functional food” is an ordinaryfood that has one or more components or ingredients incorporated into itto give a specific medical or physiological benefit, other than a purelynutritional effect.

In an embodiment, the pharmaceutical composition may be in the form ofan immediate-release, controlled-release, or sustained-release orallyadministrable composition for a pill, tablet, capsule, gelcap, lozenge,throat spray, solution, emulsion, cream, paste, gel, cough drop, ordissolvable strip. In an embodiment, the pharmaceutical composition maybe in the form of a liquid solution, liquid spray, emulsion, cream, gel,lotion, or impregnated dressing. In an embodiment, the pharmaceuticalcomposition may in the form of nasal drops, oral drops, eye drops, oraerosol trigger.

The present teachings are illustrated by the following examples.

Example 1 Preparation of Opuntia littoralis Alcohol Extract (OLAE)

Two kilos of the dried plant powder were extracted with 70% aqueousethanol four times for 8 days. The obtained greenish sticky residue wasdissolved in water, treated with excess of ethanol and filtered toremove inorganic salts and non-phenolic compounds. The alcoholic extractwas evaporated under reduced pressure at 50° C. until dry.

Example 2 Preparation of Therapeutic Composition

A lutein enriched extract preparation was prepared by mixing luteinpowder (Pure Tru Herb Private Limited) with stearic acid to provide anemulsified mixture or emulsified lutein homogenate including an amountof 1 mM to 10 mM lutein. The emulsified lutein homogenate was mixed withthe alcoholic extract (0.1 g to 1 g) of Opuntia littoralis. The mixturewas reduced by sonicating for 90 seconds using a pulse sequence thatconsisted of 25% alcoholic extract with different concentrations oflutein (Fisher Scientific, Madison, WI, USA). The emulsion was used forin vitro and in vivo analysis.

Example 3 Myeloperoxidase (MPO) Quantification

The expression of oxidative stress in lipopolysaccharide (LPS)-inducedRAW 264.7 cell lines was estimated. The activity of Myeloperoxidase(MPO), a marker of neutrophilic infiltration, and the nitrite levels inμM/mg of OLAE treated cells were determined according to the methoddescribed in Hairul et al., 2021. Briefly, lipopolysaccharide(LPS)-induced RAW cells were homogenized using lysate buffer(Invitrogen, Waltham, Massachusetts, USA). Cell-free lysate wasseparated using centrifugation. The absorbance was recorded using aspectrophotometer at 512 nm (Thermo scientific, Waltham, Massachusetts,USA). FIGS. 1A-1C show results of this analysis. MPO activity wasexpressed as units per milligram of wet tissue. One unit expresses theMPO activity needed for the conversion of 1 mM of H₂O₂ to water in 1 minat room temperature. The intensity of color modification was modifiedbased on levels of free lipid peroxidation in the samples.

Example 4 Cytotoxic Activity

Potential cytotoxicity was measured by SRB assay of the differentconcentrations of the cladode extracts of O. littoralis alcohol extractOLAE (25 μg/mL) and Lutein (2.5 μM) treatment, by the method describedby Skehan et al. (1990) using a murine macrophage cell line. FIG. 2 is agraph showing cell viability of the different concentrations of thecladode extracts of O. littoralis alcohol extract OLAE (25 μg/mL) andLutein. The intensity of color was inversely proportional to cellviability and was measured using an ELISA reader (450 nM). A plot of therelation between survival fraction and dry concentration was designed toget the survival curve of each tumor cell line after the extracttreatment.

Example 5 Nitric Oxide (NO) Quantification

The expression of oxidative stress and the nitrite levels in limb tissuewere estimated. The nitrite levels in μM/mg of tissue were determinedaccording to the method in (Khalifa et al., 2022). Briefly, theLPS-induced cells were homogenized using lysate buffer (Invitrogen,Waltham, Massachusetts, USA). Cell-free lysate was separated usingcentrifugation 8000 g rpm. The absorbance was recorded using aspectrophotometer at 512 nm (Thermo scientific, Waltham, Massachusetts,USA). MPO activity was expressed as units per milligram of wet tissue.For NO quantification, the homogenate of the above-mentioned samples wasmixed with Griess reagent, and the color intensity of the Griess reagentwas modified based on levels of free nitrates in the samples. Theresults of this analysis are summarized in FIG. 3 .

Example 6 Cell-Invasion Assay (Transwell-Assay)

Cell invasion was assessed using transwell cell culture Boyden chambers,according to the manufacturer's protocol. Gelatin coated 12-well platecell culture inserts (BD Biosciences) were used with a polyethyleneterephthalate membrane (8-μm porosity) and the inserts were incubatedfor 6 h at 37° C. Before OLAE (25 μg/mL) and Lutein (2.5 μM) treatment,100 μL of MDA cells (5×10⁴) were seeded in the upper chamber inserum-free media treated with OLAE and Lutein at the indicatedconcentrations and 700 μL of DMEM medium supplemented with 10% FBS wasadded to the lower chamber. The cells were then incubated for 24 h at37° C. Then, the remaining cells on the top surface of the membrane wereremoved using a cotton swab, and the cells on the bottom of the membranewere fixed in cold methanol (75%) for 15 min and washed with PBS threetimes. Afterward, the cells were stained with Giemsa (30%) stainingsolution and washed with PBS. Then, cells in five randomly selectedfields were counted under a light microscope at 20× objectivemagnification. The number of migrated cells was tallied by opticalmicroscopy (magnification: 200×) and manual counting. All assays wereperformed in triplicate. The results are summarized in FIG. 4 .

It is to be understood that the therapeutic composition includingphenolic compounds derived from Opuntia littoralis is not limited to thespecific embodiments described above, but encompasses any and allembodiments within the scope of the generic language of the followingclaims enabled by the embodiments described herein, or otherwise shownin the drawings or described above in terms sufficient to enable one ofordinary skill in the art to make and use the claimed subject matter.

We claim:
 1. A method of preparing a therapeutic composition includingphenolic compounds comprising: extracting a plant powder from a part ofthe Opuntia littoralis plant; combining the plant powder with water andan alcohol to provide an alcohol extract; and combining the alcoholextract with lutein to provide the therapeutic composition; wherein theplant powder is extracted from the part of the Opuntia littoralis plantwith 70% aqueous ethanol over multiple days.
 2. The method of claim 1,wherein the plant powder is obtained from a plant part of Opuntialittoralis selected from the group consisting of at least one of a fruitor cladode of the Opuntia littoralis plant.
 3. The method of claim 1,wherein about 0.1 g to about 1 g of Opuntia littoralis is combined withabout 1 mM to about 10 mM lutein.
 4. The method of claim 1, wherein thelutein is a lutein powder and is emulsified with stearic acid prior tocombination with the alcohol extract.
 5. The method of claim 1, whereinthe plant powder is extracted from the part of the Opuntia littoralisplant with 70% aqueous ethanol for 8 days.
 6. A therapeutic compositionincluding phenolic compounds derived from Opuntia littoralis, comprisingan alcoholic extract of Opuntia littoralis and lutein prepared by themethod of claim 1.